The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro

Watkins, Sarah; Borthwick, Gillian M.; Arthur, Helen M.

doi:10.1007/s11626-010-9368-1

Cited by 338 publications

(252 citation statements)

References 30 publications

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“…These phenotypes are similar to previously reported cardiomyocyte hypertrophic responses in vitro. 13,[16][17][18] TGFb1 alone also stimulated comparable hypertrophic responses to those induced by Ang II treatment (Figures 1a-c), and in a similar way to a previous report. 19 Functional TGFb1 signaling is essential for the stimulation of cardiomyocyte hypertrophy by Ang II Immunoblot experiments showed that TGFb1 and AngII stimulated phosphorylation of Smad2 in PNCM cells (Figure 2a), in a similar way to previous reports.…”

Section: Ang II and Tgfb1 Induce Similar Hypertrophic Responses In Vitrosupporting

confidence: 91%

“…2,12 We have previously shown that endothelin-1 and Ang II generated a very similar hypertrophic response in vitro. 13 Using this model we now show that Ang IIstimulated cardiomyocyte hypertrophy is dependent on TGFb receptor activity and requires the presence of TAK1, but not SMAD2/3.…”

Section: Introductionmentioning

confidence: 76%

“…13 Heart ventricles were digested with collagenase (0.4 mg ml À1 ) and pancreatin (0.6 mg ml À1 ) in 116 mM NaCl, 20 mM HEPES (pH 7.35), 0.8 mM NaH 2 PO 4 , 5.6 mM glucose, 5.4 mM KCl and 0.8 mM MgSO 4 . The recovered cells were pre-plated for 30 min on 60 mm Primaria culture dishes in plating medium (70% Dulbecco's modified Eagle's medium, 15% M199, 15% fetal calf serum, 100 units per ml penicillin and streptomycin) and non-adherent primary neonatal cardiomyocytes (PNCM) were transferred to gelatin-coated 60-mm Primaria culture dishes for 18 h, before changing to serum-free maintenance media (80% Dulbecco's modified Eagle's medium, 20% M199, 100 units per ml penicillin and streptomycin).…”

Section: Cell Culturementioning

confidence: 99%

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Angiotensin II-induced cardiomyocyte hypertrophy in vitro is TAK1-dependent and Smad2/3-independent

et al. 2011

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Cardiac hypertrophy occurs as an adaptation to hypertension but a sustained hypertrophic response can ultimately lead to heart failure. Angiotensin-II (Ang II) is released following hemodynamic overload and stimulates a cardiac hypertrophic response. AngII also increases expression of the regulatory cytokine, transforming growth factor-b1 (TGFb1), which is also implicated in the cardiac hypertrophic response and can stimulate activation of Smad2/3 as well as TGFb-activated kinase 1 (TAK1) signaling mediators. To better understand the downstream signaling events in cardiac hypertrophy, we therefore investigated activation of Smad2/3 and TAK1 signaling pathways in response to Ang II and TGFb1 using primary neonatal rat cardiomyocytes to model cardiac hypertrophic responses. Small interfering RNA (siRNA) knockdown of Smad 2/3 or TAK1 protein or addition of the TGFb type I receptor inhibitor, SB431542, were used to investigate the role of downstream mediators of TGFb signaling in the hypertrophic response. Our data revealed that TGFb1 stimulation leads to cardiomyocyte hypertrophic phenotypes that were indistinguishable from those occurring in response to Ang II. In addition, inhibition of the TGFb1 type receptor abolished Ang II-induced hypertrophic changes. Furthermore, the hypertrophic response was also prevented following siRNA knockdown of TAK1 protein, but was unaffected by knockdown of Smad2/3 proteins. We conclude that Ang II-induced cardiomyocyte hypertrophy in vitro occurs in a TAK1-dependent, but Smad-independent, manner.

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Section: Ang II and Tgfb1 Induce Similar Hypertrophic Responses In Vitrosupporting

confidence: 91%

Section: Introductionmentioning

confidence: 76%

Section: Cell Culturementioning

confidence: 99%

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Angiotensin II-induced cardiomyocyte hypertrophy in vitro is TAK1-dependent and Smad2/3-independent

et al. 2011

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“…Rat H9c2 cardiomyoblasts (American Type Culture Collection, Rockville, USA) were grown in DMEM media containing 10% heat‐inactivated FBS under 5% CO 2% and 95% air at 37°C. H9C2 served as an animal‐free alternative, sharing many physiological properties of primary cardiac cells (Watkins, Borthwick, & Arthur, 2011). Cells were transfected with MAO‐A siRNA, p53 siRNA or scramble siRNA (on‐target‐plus smart pool; Dharmacon) with DharmaFECT Duo (Dharmacon).…”

Section: Methodsmentioning

confidence: 99%

Monoamine oxidase‐A is a novel driver of stress‐induced premature senescence through inhibition of parkin‐mediated mitophagy

et al. 2018

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Cellular senescence, the irreversible cell cycle arrest observed in somatic cells, is an important driver of age‐associated diseases. Mitochondria have been implicated in the process of senescence, primarily because they are both sources and targets of reactive oxygen species (ROS). In the heart, oxidative stress contributes to pathological cardiac ageing, but the mechanisms underlying ROS production are still not completely understood. The mitochondrial enzyme monoamine oxidase‐A (MAO‐A) is a relevant source of ROS in the heart through the formation of H2O2 derived from the degradation of its main substrates, norepinephrine (NE) and serotonin. However, the potential link between MAO‐A and senescence has not been previously investigated. Using cardiomyoblasts and primary cardiomyocytes, we demonstrate that chronic MAO‐A activation mediated by synthetic (tyramine) and physiological (NE) substrates induces ROS‐dependent DNA damage response, activation of cyclin‐dependent kinase inhibitors p21cip, p16ink4a, and p15ink4b and typical features of senescence such as cell flattening and SA‐β‐gal activity. Moreover, we observe that ROS produced by MAO‐A lead to the accumulation of p53 in the cytosol where it inhibits parkin, an important regulator of mitophagy, resulting in mitochondrial dysfunction. Additionally, we show that the mTOR kinase contributes to mitophagy dysfunction by enhancing p53 cytoplasmic accumulation. Importantly, restoration of mitophagy, either by overexpression of parkin or inhibition of mTOR, prevents mitochondrial dysfunction and induction of senescence. Altogether, our data demonstrate a novel link between MAO‐A and senescence in cardiomyocytes and provides mechanistic insights into the potential role of MAO‐dependent oxidative stress in age‐related pathologies.

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“…First, for the assessment of cell viability as a fundamental evaluation of cellular toxicity, we used H9c2 cells, which is a frequently used cardiac in vitro cellular model. The H9c2 cell line is derived from embryonic rat heart tissue and has been validated as a reliable model for its ability to mimic development and disease states [20]. Using this model, we uncovered the potential for DGA to decrease cell viability.…”

Section: Discussionmentioning

confidence: 99%

Cardiotoxicity testing of diglycolic acid using In Vitro and In Vivo models

Calvert¹,

Mossoba²,

Bailey³

et al. 2017

J Toxicol Health

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Background: Renal and hepatotoxicity of diglycolic acid (DGA) has been described with in vitro cellular models as well as in vivo animal and human systems. The possibility of DGA being toxic to other organs, such as the heart, has not been well investigated. A human case report identified the heart as a potential target organ of DGA, but an in-house in vivo rat study neither found gross nor microscopic pathological changes following repeated oral DGA exposure. Methods: To better understand the potential of DGA to adversely affect the heart, we focused our current study on evaluating cardiac effects of DGA by treating rat H9c2 cardiomyocytes and human induced pluripotent stem cells (iPSCs) that were differentiated into beating cardiomyocytes (iCells) with increasing concentrations of DGA in cell culture media. We investigated the effects, mechanisms, and value of our in vitro cellular cardiac models. Results: We measured mild effects of DGA on cytotoxicity and the cellular production of reactive oxygen species in H9c2 cells. Cardiomyocyte beat rate (BPM) was also mildly affected by transient treatment with DGA. Conclusions: Our study indicates that DGA cardiotoxicity in vitro correlates with the preliminary findings in our in vivo study where rats treated with daily doses of up to 300 mg/kg of DGA orally for 5 days did not show any major signs of abnormal cardiac pathology.

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The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro

Cited by 338 publications

References 30 publications

Angiotensin II-induced cardiomyocyte hypertrophy in vitro is TAK1-dependent and Smad2/3-independent

Angiotensin II-induced cardiomyocyte hypertrophy in vitro is TAK1-dependent and Smad2/3-independent

Monoamine oxidase‐A is a novel driver of stress‐induced premature senescence through inhibition of parkin‐mediated mitophagy

Cardiotoxicity testing of diglycolic acid using In Vitro and In Vivo models

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